Sp1 phosphorylation is involved in myelin basic protein gene transcription.

Myelin basic protein (MBP), which helps form compact myelin sheets, is a major protein expressed during oligodendrocyte (OL) differentiation. Myelin basic protein expression is regulated mainly at the transcriptional level. Previous studies showed that the transcription factor Sp1 can activate the MBP promoter. Data from the laboratory also indicate that Sp1 is expressed highly in both growing and differentiated cells. Because this is true, we wanted to understand how Sp1 activity is regulated such that it increases MBP gene transcription only in differentiating cells. Phosphorylation is one major way to regulate transcription factor activity. Our results show that there is more Sp1 binding to the MBP promoter in differentiating OLs. Sp1 is also more phosphorylated in differentiating OLs than in precursor cells. Using inhibitors of different pathways, we found that the protein kinase C (PKC) modulator phorbol 12-myristate 13-acetate (PMA) can increase Sp1 phosphorylation when the cells are treated for 1 hr and can decrease Sp1 phosphorylation with long treatment (12 hr). The increased phosphorylation of Sp1 induced by PMA in short treatments could be abolished by the extracellular signal-regulated kinases (ERK) pathway inhibitor PD98059. This indicates that PKC phosphorylates Sp1 through the ERK pathway. Mutation of Sp1 threonines 453 and 739, which are phosphorylated by ERK, decreased MBP transcriptional activity. Furthermore, we found that PKC regulates Sp1 phosphorylation only in differentiating OLs. In conclusion, our results indicate that, in OLs, Sp1 phosphorylation can be regulated by PKC-ERK pathways. This phosphorylation is important for MBP transcription and oligodendrocyte differentiation.

In utero PCP exposure alters oligodendrocyte differentiation and myelination in developing rat frontal cortex.

Several neurodevelopmental disorders, including schizophrenia, autism, ADD/ADHD and dyslexia are believed to originate during gestation and involve white matter abnormalities. Modulation of glutamate environments and glutamate receptors has also been implicated in alteration of oligodendrocytes, the myelin forming cells of the CNS. To begin to understand how modulation of the glutamate system affects the maturation of oligodendrocytes, developing rats were subjected to prenatal blockade of the NMDA receptor with phencyclidine (PCP). Oligodendrocyte development and differentiation were then examined postnatally by measuring markers for early, middle and late stage cells. The results indicate that, while the level of marker proteins for neurons and astrocytes remains the same, early oligodendrocyte progenitor cell markers are decreased in rat brains prenatally exposed to PCP. Labeling of cells of intermediate, immature cell stages is elevated. Late stage markers for myelinating oligodendrocytes are subsequently decreased. These data suggest that prenatal NMDA receptor blockade reduces the level of progenitors and that the surviving cells are arrested at an immature stage. This premature arrest appears to result in fewer fully differentiated, mature oligodendrocytes that are capable of producing myelin. These results have interesting implications for the role of glutamate and glutamate receptors in white matter abnormalities in neurodevelopmental disorders.

Cap-independent translation through the p27 5'-UTR.

Several recent publications have explored cap-independent translation through an internal ribosome entry site (IRES) in the 5'-UTR of the mRNA encoding the cyclin-dependent kinase inhibitor p27. The major experimental tool used in these reports was the use of bicistronic reporter constructs in which the 5'-UTR was inserted between the upstream and downstream cistrons. None of these reports has completely ruled out the possibility that the 5'-UTR has either cryptic promoter activity or a cryptic splice acceptor site. Either of these possibilities could result in expression of a monocistronic mRNA encoding the downstream cistron and false identification of an IRES. Indeed, Liu et al. recently published data suggesting that the p27 5'-UTR harbors cryptic promoter activity which accounts for its putative IRES activity. In this report, we have explored this potential problem further using promoterless bicistronic constructs coupled with RNase protection assays, siRNA knockdown of individual cistrons, RT-PCR to detect mRNA encoded by the bicistronic reporter with high sensitivity, direct transfection of bicistronic mRNAs, and insertion of an iron response element into the bicistronic reporter. The results do not support the conclusion that the p27 5'-UTR has significant functional promoter activity or cryptic splice sites, but rather that it is able to support cap-independent initiation of translation.

Galectin-4 is involved in p27-mediated activation of the myelin basic protein promoter.

Our previous studies have found that expression of p27 in oligodendrocytes enhances myelin basic protein (MBP) gene expression through a mechanism that involves the transcription factor Sp1. In this study we show that this activation only requires the N-terminal 45 amino acids of p27 containing a functional cyclin-binding motif. In an effort to identify other cofactors that are involved in the p27-mediated activation of MBP gene expression, a yeast two-hybrid assay was performed using an N-terminal truncated p27 and a mouse embryo cDNA library. Galectin-4 was found to interact with p27 in the yeast two-hybrid assay. This novel interaction was also confirmed using a glutathione-S-transferase interaction assay and immunoprecipitation assays. Expression of galectin-4 in primary oligodendrocytes was confirmed by western blot. Additionally, the MBP promoter could be activated by expression of galectin-4 in CG4 oligodendrocytes, similar to the effects of increased p27 levels. We also show that Sp1 and galectin-4 interact in cells, while a complex of all three proteins could not be found. We conclude that galectin-4 is involved in the p27-mediated activation of the MBP gene, possibly through modulation of the glycosylation status of the transcription factor Sp1.

Stage-specific expression of myelin basic protein in oligodendrocytes involves Nkx2.2-mediated repression that is relieved by the Sp1 transcription factor.

The homeodomain-containing protein Nkx2.2 is critical for the development of oligodendrocyte lineage cells, but the target genes of Nkx2.2 regulation have not been identified. In the present study, we found that the myelin basic protein gene is one of the genes that is regulated by Nkx2.2. Expression of Nkx2.2 represses the expression of myelin basic protein in oligodendrocyte progenitors. Two regulatory elements in the myelin basic protein promoter were identified and found to interact with Nkx2.2 in vitro. Despite their sequence divergence, both sites were involved in the Nkx2.2-mediated repression of the myelin basic protein promoter. Binding of Nkx2.2 also blocked and disrupted the binding of the transcriptional activator Puralpha to the myelin basic protein promoter. Additionally Nkx2.2 recruited a histone deacetylase 1-mSin3A complex to the myelin basic protein promoter. We also found that the transcription factor Sp1 was able to compete off the binding of Nkx2.2 to its consensus binding site in vitro and reversed the repressive effect of Nkx2.2 in vivo. Our data revealed a novel role for Nkx2.2 in preventing the precocious expression of myelin basic protein in immature oligodendrocytes. Based on this study and our previous reports, a model for myelin basic protein gene control is proposed.

Sox10 acts as a tissue-specific transcription factor enhancing activation of the myelin basic protein gene promoter by p27Kip1 and Sp1.

Myelin basic protein (MBP) is one of the major components of the myelin sheath that insulates axons. In the central nervous system, MBP is synthesized by differentiating oligodendrocytes. The expression of MBP in oligodendrocytes is regulated mainly at the transcriptional level. The Sp1 family of transcription factors has been shown to be important in the regulation of many genes. Binding of Sp1 to the GC box in the proximal MBP promoter has been shown to be indispensable for the activation of MBP gene expression. Previous results from our laboratory have shown that the increase in p27Kip1 that accompanies oligodendrocyte differentiation is paralleled by an increase in Sp1. We also have shown that the increase in MBP expression resulting from elevated p27Kip1 levels is mediated through Sp1 and that this effect occurs specifically in oligodendrocytes. In this study, we found that increased expression of p27Kip1 together with the nervous-system-specific transcription factor Sox10 can activate the MBP promoter even in nonoligodendrocyte cells. This indicates that Sox10 confers cell type specificity on the expression of MBP. Both Sp1 and Sox10 can enhance MBP promoter activity when expressed alone. Cotransfection of plasmids encoding Sp1 and Sox10 induces increased activation of the MBP promoter over expression of either transcription factor alone. This effect is not limited to oligodendrocyte cell lines, in that Sp1 and Sox10 can also synergistically activate the MBP promoter when expressed in NIH3T3 fibroblasts. Mutation of the Sp1 binding sites in the MBP promoter eliminates Sox10 stimulated activation, suggesting that the MBP promoter is activated, at least in part, through protein-protein interactions between Sp1 and Sox10.

Regulation of p27(Kip1) by intracellular iron levels.

Enhanced intracellular iron levels are essential for proliferation of mammalian cells. If cells have entered S phase when iron is limiting, an adequate supply of deoxynucleotides cannot be maintained and the cells arrest with incompletely replicated DNA. In contrast, proliferating cells that are not in S phase, but have low iron pools, arrest in late G1. In this report the mechanism of iron-dependent G1 arrest in normal fibroblasts was investigated. Cells were synchronized in G0 by contact inhibition and serum deprivation. Addition of serum caused the cells to re-enter the cell cycle and enter S phase. However, if the cells were also treated with the iron chelator deferoxamine, S phase entry was blocked. This corresponded to elevated levels of the cyclin dependent kinase inhibitor p27(Kip1) and inhibition of CDK2 activity. Expression of other cell cycle regulatory proteins was not affected, including the induction of cyclins D1 and E. When the quiescent serum starved cells were supplemented with a readily usable form of iron in the absence of serum or any other growth factors, a significant population of the cells entered S phase. This was associated with downregulation of p27(Kip1) and increased CDK2 activity. Using an IPTG-responsive construct to artificially raise p27(Kip1) levels blocked the ability of iron supplementation to promote S phase entry. Thus it appears that p27(Kip1) is a mediator of G1 arrest in iron depleted Swiss 3T3 fibroblasts. We propose that this is part of an iron-sensitive checkpoint that functions to ensure that cells have sufficient iron pools to support DNA synthesis prior to entry into S phase.

Cyclin dependent kinase inhibitor p27(Kip1) is upregulated by hypoxia via an ARNT dependent pathway.

Expression of cyclin dependent kinase (Cdk) inhibitor p27(Kip1), which blocks cell cycle progression from G(1) to S phase, can be regulated via multiple mechanisms including transcription, protein degradation, and translation. Recently, it was shown that p27(Kip1) plays an important role in the cellular response to hypoxia. However, the mechanisms involved in the hypoxia-induced regulation of p27(Kip1) expression are still not clear. In this study, we compare the expression of p27(Kip1) in two related murine hepatoma cell lines, Hepa-1 and c4. Hepa-1 produces functional aryl hydrocarbon receptor nuclear translocator (ARNT). c4 cells are derived from Hepa-1, but are ARNT deficient. Interestingly, we observed cell line-dependent effects of hypoxia on the expression of p27(Kip1). The level of p27(Kip1) protein in Hepa-1 cells is enhanced by hypoxia, but is reduced by hypoxia in c4 cells. Further investigation demonstrated that hypoxia-induced, ARNT-mediated, transactivation of the p27(Kip1) gene in Hepa-1 cells is responsible for the increase in p27(Kip1) protein. Once c4 cells were stably transfected with the wild type ARNT gene, a hypoxia-induced increase in p27(Kip1) mRNA was observed and reduction of p27(Kip1) protein caused by hypoxia was blocked. Hence, our data indicate that ARNT is involved in transcriptional upregulation of the p27(Kip1) gene under hypoxic conditions.

Cloning and characterization of the rat myelin basic protein gene promoter.

Expression of myelin basic protein in differentiating oligodendrocytes is mainly regulated at the transcriptional level. To better understand the regulation of myelin basic protein gene expression in mammalian cells, we cloned and characterized the rat myelin basic protein promoter by a genome walking technique. Extensive sequence homology has been found among mouse, rat and human MBP promoters. Alignment of the proximal core promoter of mouse and rat reveals highly conserved cis-elements that are important for regulating myelin basic protein gene transcription. One major transcription start site along with two minor sites have been identified in both mouse and rat myelin basic protein gene promoters using RNA ligase-mediated rapid amplification of 5' cDNA ends. The amplified rat myelin basic protein promoter was cloned into a luciferase reporter construct. Transient transfection experiments show that both mouse and rat myelin basic protein promoters yield increased expression when oligodendrocytes differentiate. The sequence and characterization of the rat MBP promoter provide a useful tool to investigate MBP gene regulation in mammalian cells.

The Sp1 family of transcription factors is involved in p27(Kip1)-mediated activation of myelin basic protein gene expression.

p27(Kip1) levels increase in many cells as they leave the cell cycle and begin to differentiate. The increase in p27(Kip1) levels generally precedes the expression of differentiation-specific genes. Previous studies from our laboratory showed that the overexpression of p27(Kip1) enhances myelin basic protein (MBP) promoter activity. This activation is specific to p27(Kip1). Additionally, inhibition of cyclin-dependent kinase activity alone is not sufficient to increase MBP expression. In this study, we focused on understanding how p27(Kip1) can activate gene transcription by using the MBP gene in oligodendrocytes as a model. We show that the enhancement of MBP promoter activity by p27(Kip1) is mediated by a proximal region of the MBP promoter that contains a conserved GC box binding sequence. This sequence binds transcription factors Sp1 and Sp3. Increased expression of p27(Kip1) increases the level of Sp1 promoter binding to the GC box but does not change the level of Sp3 binding. The binding of Sp1 to this element activates the MBP promoter. p27(Kip1) leads to increased Sp1 binding through a decrease in Sp1 protein turnover. Enhancement of MBP promoter activity by an increase in the level of p27(Kip1) involves a novel mechanism that is mediated through the stabilization and binding of transcription factor Sp1.

Expression of constitutively active 4EBP-1 enhances p27Kip1 expression and inhibits proliferation of MCF7 breast cancer cells.

BACKGROUND: Eukaryotic initiation factor 4E (eIF4E) is essential for cap-dependent initiation of translation. Cell proliferation is associated with increased activity of eIF4E and elevated expression of eIF4E leads to tumorigenic transformation. Many tumors express very high levels of eIF4E and this may be a critical factor in progression of the disease. In contrast, overexpression of 4EBP, an inhibitor of eIF4E, leads to cell cycle arrest and phenotypic reversion of some transformed cells. RESULTS: A constitutively active form of 4EBP-1 was inducibly expressed in the human breast cancer cell line MCF7. Induction of constitutively active 4EBP-1 led to cell cycle arrest. This was not associated with a general inhibition of protein synthesis but rather with changes in specific cell cycle regulatory proteins. Cyclin D1 was downregulated while levels of the CDK inhibitor p27Kip1 were increased. The levels of cyclin E and CDK2 were unaffected but the activity of CDK2 was significantly reduced due to increased association with p27Kip1. The increase in p27Kip1 did not reflect changes in p27Kip1 mRNA or degradation rates. Rather, it was associated with enhanced synthesis of the protein, even though 4EBP-1 is expected to inhibit translation. This could be explained, at least in part, by the ability of the p27Kip1 5'-UTR to mediate cap-independent translation, which was also enhanced by expression of constitutively active 4EBP-1. CONCLUSIONS: Expression of active 4EBP-1 in MCF7 leads to cell cycle arrest which is associated with downregulation of cyclin D1 and upregulation of p27Kip1. Upregulation of p27Kip1reflects increased synthesis which corresponds to enhanced cap-independent translation through the 5'-UTR of the p27Kip1 mRNA.

Phosphatidylinositol-3 kinase p85 enhances expression from the myelin basic protein promoter in oligodendrocytes.

Phosphatidylinositol-3 kinase (PI3K) is a family of enzymes that phosphorylates the D3 position of phosphoinositides in membranes which can then act as a second messenger and affect many essential cellular processes such as survival, proliferation and differentiation. Class IA PI3K is composed of two subunits: a regulatory subunit, p85, and a catalytic subunit, p110. The p85 subunit is composed of several adapter domains which, upon interaction with the appropriate molecules, transmit the signal to activate p110. We have used the spontaneously immortalized oligodendrocyte cell line, CG4, to examine the role of PI3K in maturation of the oligodendrocyte. We show that overexpression of the p85 subunit enhances expression of myelin basic protein (MBP) upon differentiation of CG4 cells and primary oligodendrocytes. In experiments in CG4 cells, neither cotransfection with the tumor suppressor PTEN, which dephosphorylates the D3 position of phosphoinositides, nor inhibition of PI3K activity with wortmannin mimics this effect. Further, we have shown that this effect is dependent on the coexpression of the two SH2 domains within p85. Thus, the p85-mediated enhancement of MBP promoter activity in oligodendrocytes appears to be independent of PI3K activity and dependent on the adapter functions of the p85 subunit's SH2 domains.

Cyclic AMP inducibility of the myelin basic protein gene promoter requires the NF1 site.

In the central nervous system oligodendrocyte differentiation is accompanied by the activation of a specific transcriptional program responsible for the synthesis of myelin genes. One of the signals leading to the expression of myelin components, such as the myelin basic protein (MBP) gene is cyclic AMP (cAMP). Previous work using a cell line in which the endogenous MBP gene can be induced by increased cAMP levels (D6P2T) showed that the region of the MBP gene that was required for induction of the gene by cAMP lay between -248 and -105 in the 5' flanking region. This region contains numerous transcription factor binding sites, including sites for NF1, Sp1, and MEBA. In order to determine if the NF1 site itself was specifically responsible for the cAMP responsiveness of the MBP promoter, stably transfected cells carrying MBP promoter deletion constructs were used. Deletion of just the NF1 site caused loss of responsiveness to cAMP levels. Furthermore, site-specific mutations in the NF1 site that interfere with NF1 protein binding, in the context of the full length promoter, abolished cAMP responsiveness and caused derepression of the promoter. Analysis of protein binding to the NF1 site showed that the mutation resulted in loss of binding to the site and that the proteins binding at the site are modified in the presence of cAMP elevating agents. These results demonstrate that the NF1 site is indispensable for cAMP responsiveness of the MBP promoter and, together with other DNA elements, plays a role in controlling MBP gene expression.

p27(Kip1) enhances myelin basic protein gene promoter activity.

The process of oligodendrocyte differentiation is a complex event that requires cell cycle withdrawal, followed by the activation of a specific transcriptional program responsible for the synthesis of myelin genes. Because growth arrest precedes differentiation, we sought to investigate the role of cell cycle molecules in the activation of myelin gene promoters. We hypothesized that the cell cycle inhibitor p27(Kip1), which is primarily responsible for arresting proliferating oligodendrocyte progenitors, may be involved in the transcriptional regulation of myelin genes. In agreement with this hypothesis, overexpression of p27(Kip1) in the CG4 cell line, but not in 3T3 fibroblasts, enhances the expression of luciferase driven by the myelin basic protein (MBP) promoter. Interestingly, this effect is specific for p27(Kip1); overexpression of other cell cycle inhibitors had no effect. Additionally, this effect is independent of halting the cell cycle; treatment with the cell cycle blocker roscovitine did not affect MBP promoter usage. We conclude that p27(Kip1) contributes to oligodendrocyte differentiation by regulating transcription of the MBP gene.